Ahead of print online version Uncinaria sanguinis sp. n. (Nematoda

Ahead of print online version
Folia Parasitologica 61 [3]: 255–265, 2014
© Institute of Parasitology, Biology Centre ASCR
http://folia.paru.cas.cz/
ISSN 0015-5683 (print), ISSN 1803-6465 (online)
doi: 10.14411/fp.2014.037
Uncinaria sanguinis sp. n. (Nematoda: Ancylostomatidae)
from the endangered Australian sea lion, Neophoca cinerea
(Carnivora: Otariidae)
Alan D. Marcus, Damien P. Higgins, Jan Šlapeta and Rachael Gray
Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia
Abstract: This study investigates the identity of hookworms parasitising the Australian sea lion, Neophoca cinerea (Péron), from
three colonies in South Australia, Australia. The Australian sea lion is at risk of extinction because its population is small and genetically fragmented. Using morphological and molecular techniques, we describe a single novel species, Uncinaria sanguinis sp. n.
(Nematoda: Ancylostomatidae). The new species is most similar to hookworms also parasitic in otariid hosts, Uncinaria lucasi Stiles,
1901 and Uncinaria hamiltoni Baylis, 1933. Comparative morphometrics offered limited utility for distinguishing between species
within this genus whilst morphological features and differences in nuclear ribosomal DNA sequences delineated U. sanguinis sp.
n. from named congeners. Male specimens of U. sanguinis sp. n. differ from U. lucasi and U. hamiltoni by relatively shorter anterolateral and externodorsal rays, respectively, and from other congeners by the relative lengths and angulations of bursal rays, and
in the shape of the spicules. Female specimens of U. sanguinis sp. n. are differentiated from Uncinaria spp. parasitic in terrestrial
mammals by differences in vulval anatomy and the larger size of their eggs, although are morphologically indistinguishable from
U. lucasi and U. hamiltoni. Molecular techniques clearly delimited U. sanguinis sp. n. as a distinct novel species. Obtaining baseline
data on the parasites of wildlife hosts is important for the investigation of disease and the effective implementation and monitoring
of conservation management.
Keywords: new species, hookworms, conservation, phylogeny, pinnipeds
This article contains supporting information (Tables S1, S2) online at http://folia.paru.cas.cz/suppl/2014-3-255.pdf
Hookworms of the genus Uncinaria Frölich, 1789
(Nematoda: Ancylostomatidae) are haematophagous
parasitic nematodes of the small intestine. Eleven species
have been described in carnivores (Mammalia: Carnivora) and three additional species described in rodents (Rodentia), tenrecs (Afrosoricida) and treeshrews (Scandentia). The validity of several species has been disputed as
a result of unproven host specificity and the questionable
significance of some morphological differences (Ransom
1924, Baylis 1933, Olsen 1968, Beveridge 1980). Molecular techniques coupled with morphological analysis can
reduce the uncertainty of species delimitation and description (Pérez-Ponce de León and Nadler 2010). Baseline
knowledge of species identity has significant implications
for the management of parasitic diseases and conservation of the host themselves (Thompson et al. 2010).
One of the long-standing parasitological uncertainties
is the number of distinct Uncinaria species parasitising
pinnipeds (Carnivora) (George-Nascimento et al. 1992,
Nadler et al. 2000). Parasites in the genus Uncinaria
have been reported from eleven otariid (eared seals) and
five phocid (earless seals) hosts (Dailey 2001, Lyons et
al. 2011a, Brock et al. 2013, Nadler et al. 2013). Only
two species have been described and named, Uncinaria
lucasi Stiles, 1901 from the northern fur seal, Callorhinus ursinus (Linnaeus), and Uncinaria hamiltoni Baylis,
1933 from the South American sea lion, Otaria byronia de
Blainville; syn. Otaria flavescens (Shaw). However, the
existence of greater species diversity has been recently
recognised using molecular techniques (Nadler et al.
2000, Lyons et al. 2011b, Nadler et al. 2013, Ramos et al.
2013). Additionally, Nadler et al. (2013) demonstrated reduced host-specificity, showing that U. lucasi also parasitises Steller sea lion, Eumetopias jubatus (Schreber), and
U. hamiltoni also parasitises the South American fur seal,
Arctophoca australis (Zimmerman). The taxonomic identity of specimens from other pinniped hosts is unresolved.
In Australian waters, hookworms from the Australian
sea lion, Neophoca cinerea (Péron), Australian fur seal,
Arctocephalus pusillus doriferus (Wood Jones), and New
Zealand fur seal, Arctophoca australis forsteri (Lesson),
are traditionally considered closely related and morpho-
Address for correspondence: R. Gray, Faculty of Veterinary Science, The University of Sydney, McMaster Bldg B14, Sydney, New South Wales
2006, Australia. Phone: +61 2 9351 2643; Fax: +61 2 9351 7421; E-mail: [email protected]
255
Ahead of print online version
logically similar to U. hamiltoni (Norman 1994, Ramos
et al. 2013). Ramos et al. (2013) showed that Uncinaria
spp. from Australian pinnipeds are molecularly distinct
from North American pinniped hookworms and that the
Australian sea lion and New Zealand fur seal may share
a single species of Uncinaria.
The aim of the present study was to address whether
a single novel species of hookworm parasitises the Australian sea lion. The Australian sea lion is classified as
endangered in the IUCN Red List of Threatened Species due to its small, genetically fragmented population,
population declines at some colonies, and the risk of extinction from fishery by-catch (Goldsworthy and Gales
2008, McIntosh et al. 2012). We investigated the utility of
quantitative morphometrics to delineate Uncinaria species by assessing intra- and inter-host parasite variation
and determining the effect of host age on parasite morphometrics. Subsequently, we used molecular techniques
to examine the phylogenetic relationships of Uncinaria
species. The outcome is a description of a new Uncinaria
species.
MATERIALS AND METHODS
Sample collection
All samples were collected from Australian sea lion pups.
Necropsies were undertaken on pups found dead at Seal Bay,
Kangaroo Island (n = 84), Dangerous Reef, Spencer Gulf
(n = 32), and South Page Island, Backstairs Passage (n = 1), in
South Australia during 2009–2013 as a part of ongoing investigations into the pathogenesis and epidemiology of hookworm infection in the Australian sea lion. A sample of hookworm specimens was collected from the small intestine from fresh carcasses
or from frozen-thawed pups and stored in 70% ethanol at -20 °C;
the remainder of the intestinal content was stored in 10% neutral
buffered formalin. Daily observations of marked and unmarked
pups at Seal Bay in 2012 facilitated the collection of hookworm
specimens from dead pups of known-age (8–101 days, n = 12).
All samples were collected under Government of South Australia Department of Environment, Water and Natural Resources
Wildlife Ethics Committee approvals (3–2008 and 3–2011) and
Scientific Research Permits (A25008/4–8).
Morphological study
Individual hookworms were examined in temporary glass
slide mounts using 4, 10, 20, and 40× objectives of an Olympus BX60 microscope equipped with differential interference
contrast (Olympus, Sydney, Australia) and photographed with
a ProgRes CFscan camera (Jenoptik, Jena, Germany) or DP80
camera (Olympus). Voucher specimens preserved with 70%
ethanol were cleared and mounted with lactophenol (Rep 1963).
Hookworm vouchers used for molecular studies were mounted
in 70% ethanol. Measurements recorded with an ocular micrometer were body length, maximum body width (measured
at approximately mid-specimen), buccal capsule length, buccal
capsule width, teeth height, oesophageal length, and maximum
oesophageal width. Additional measurements recorded from
male specimens included spicule length and gubernaculum
length, and from female specimens the distance from the vulva
to tail tip, tail length, and the average length and width of three
256
eggs per female. Measurements for each feature were not obtained for every specimen due to differences in preservation or
clarity. Additional morphological observations were performed
with specimens cleared and mounted in glycerol. Specimens
obtained from pups with hookworm eggs in their faeces were
considered mature, confirmed by the presence of eggs in female
hookworms. Measurements given in-text are mean with standard deviation, followed in parentheses by the range and sample
size. Values in brackets are measurements for the holotype male
or allotype female, as indicated. All measurements are in micrometres, unless otherwise stated.
Z-stack images were created using cellSens Dimensions
1.8.1 (Olympus). All images were imported into Adobe Photoshop CS6 (Adobe Systems, San Jose, USA) and multiple images were combined to illustrate features spanning greater than
a single field-of-view. Images were converted to greyscale and
adjustment layers for levels and brightness/contrast were used
to optimise the appearance of investigated features. Individual
specimens were isolated from the background using layer masks
and presented on a white background. Line drawings accompany photomicrographs to illustrate characteristic features. The introduction of the new hookworm species name followed generic
rules for describing a new (parasite) species (Šlapeta 2013).
Statistical analysis
Descriptive statistics were used to assess the intra- and interhost morphometric differences between hookworm specimens.
The effect of host age on the body length of both male and female hookworms, and on spicule length for male specimens,
was assessed with a linear fixed effects model using REML
in GenStat 16.1 (VSN International, Hemel Hempstead, UK).
Model assumptions were checked by visually assessing the
fitted-value plots of residuals for homogeneity of variance and
the histograms of residuals for approximately normal distributions. Predicted means were compared using Fisher’s Protected
LSD (α = 0.05). We focused on these features as they may be
objectively measured, are unlikely to be substantially influenced
by specimen preparation, and are considered important for species discrimination (Baylis 1933, Rep 1963, Nadler et al. 2000).
Morphological identification
The identification of examined specimens was attempted using taxonomic keys and species descriptions (Baylis 1933, 1947,
Maplestone 1939, Chabaud et al. 1964, Olsen 1968, Chabaud
and Durette-Desset 1975, Beveridge 1980, Hasegawa 1989, Lichtenfels 2009, Willmott and Chabaud 2009).
Molecular characterisation
An approximately 4 mm mid-body section was excised aseptically using a sterile scalpel blade from individual hookworms
collected from Seal Bay (n = 10; 10 hosts), Dangerous Reef
(n = 10; 8 hosts) and South Page Island (n = 1). The anterior and
posterior ends were retained as voucher specimens. The midsection of each hookworm was air dried prior to DNA extraction.
DNA was extracted from mid-body sections using the standard
protocol of the Isolate II Genomic DNA Kit (Bioline, Sydney,
Australia).
Two regions of nuclear ribosomal DNA (rDNA) encompassing the internal transcribed spacers (ITS1 and ITS2) and a partial sequence of the 28S rDNA were amplified by PCR using
primers No. 93/No. 94 and No. 527/No. 532, respectively (Nadler et al. 2000). PCR was performed using 15 µl MyTaq Red
Mix (Bioline), 5 pmol of each primer, and 2 µl template DNA in
Ahead of print online version
Marcus et al.: Uncinaria sanguinis sp. n. from Neophoca cinerea
a total volume of 30 µl. Cycling parameters consisted of an initial denaturation at 94 °C for 5 min, followed by 30 or 35 cycles
of 94 °C for 15 s, 54 °C for 15 s, 72 °C for 15 s, and a final extension at 72 °C for 2 min. Sequence polymorphism was verified by
PCR with an alternate polymerase master mix, EconoTaq PLUS
GREEN (Lucigen, Middleton, USA), using the same parameters
described above. Successful amplification was confirmed by gel
electrophoresis using a 1.5% agarose gel with GelRed (Biotium,
Hayward, USA). Amplicons were bidirectionally sequenced using PCR primers at Macrogen Inc. (Seoul, South Korea). Sequences were assembled and analysed with CLC Main Workbench 6.8.1 (CLC bio, Aarhus, Denmark).
Obtained sequences were aligned with hookworm sequences from the otariid hosts Australian fur seal (ITS: HE962177;
28S rDNA: HQ261929), New Zealand fur seal (ITS region
only: HE962175), New Zealand sea lion, Phocarctos hookeri (Gray) (HQ262095; HQ261983), South American sea lion
(HQ262119; HQ262006), South American fur seal (HQ262102;
HQ261989), Steller sea lion (HQ262131; HQ262018), northern
fur seal (AF217890; AF257730), and California sea lion, Zalophus californianus (Lesson) (AF217889; AF257724), and
from the phocid hosts southern elephant seal, Mirounga leonina
(Linnaeus) (HE962181; HQ262009), and Mediterranean monk
seal, Monachus monachus Hermann (HQ262065; HQ261951).
An Australian sea lion hookworm sequence (ITS region only:
HE962176), collected from Dangerous Reef in 1994 (Ramos
et al. 2013), was included for historical comparison. Uncinaria
stenocephala Railliet, 1884 (AF194145; AF257712) from the
Arctic fox, Alopex lagopus (Linnaeus), and Uncinaria felidis
Maplestone, 1939 (Shimono et al. 2012; ITS2 only) from the
Tsushima leopard cat, Prionailurus bengalensis euptilura (Elliott), were included for outgroup comparisons. The numbering of
sequence residues is according to rDNA unit of Caenorhabditis
elegans (Maupas, 1900) (X03680).
Multiple sequence alignment was constructed using ClustalW 1.83 (Thompson et al. 1994). Comparisons were performed for the ITS1, ITS2 and 28S rDNA regions to determine
percentage sequence similarity (uncorrected p-distance) using
CLC Main Workbench. Phylogenetic analyses were performed
using a concatenated DNA dataset. The best-fit model of DNA
evolution was selected based on the lowest Bayesian Information Criterion using MEGA 5.2.1 (Tamura et al. 2011). Bayesian trees were constructed using MrBayes 3.2.2 (Ronquist et al.
2012). The parameters were set to Kimura-2 parameter model
and run for 2 000 000 generations with sampling every 200 generations with four chains (temperature set to 0.2). The analysis
was run beyond convergence and run multiple times.
The consensus tree was constructed and posterior probabilities calculated from the dataset from which the first 500 000 generations were discarded. Maximum Parsimony and Maximum
Likelihood trees were calculated using MEGA 5.2.1 (Tamura
et al. 2011). The Maximum Parsimony tree was obtained using
the Subtree-Pruning-Regrafting algorithm with search level 1 in
which the initial trees were obtained by the random addition
of sequences (10 replicates). The initial Maximum Likelihood
search was started from a tree reconstructed using the Neighbour-Joining method with distances estimated using the Maximum Composite Likelihood approach. Bootstrap support was
calculated from 1 000 replicates. A schematic tree of the phylogeny demonstrating character state changes was constructed
from the majority consensus of the three phylogenetic analyses.
RESULTS
Morphological and molecular data of nematodes recovered from the Australian sea lion have revealed an undescribed hookworm species. Our current investigation
forms the basis for a formal description of a new parasite
species belonging to the genus Uncinaria.
Uncinaria sanguinis sp. n.
Figs. 1–14
Description: Small, translucent, white nematodes with
occasional dark-red intestinal contents. Sexual dimorphism evident in mature specimens (Figs. 1, 2). Anterior
extremity bent dorsally (Figs. 4, 6). Ventricose-shaped
oral opening armed with paired anterior and posterior
cutting plates (Figs. 3–6). Four small submedian papillae present around oral opening (Fig. 5), amphids not
observed. Buccal capsule large and globular with continuous walls. Dorsal gutter present. Bilateral teeth with
variably shaped tips arise ventrally, anterior to the annular thickening of buccal capsule. Oesophagus elongate,
posteriorly clavate. Paired lateral deirids, small, present
in mid-oesophageal region (Fig. 6). Nerve ring in midoesophageal region (Fig. 6). Excretory pore opens ventrally, in mid-oesophageal region (Fig. 6).
Male (description based on 116 specimens; for detailed
morphometric data – see Tables 1, 2): Total body length
9.1 ± 1.7 (3.8–11.5; 46) [8.8] mm; juvenile (8–14 days)
7.2 ± 1.7 (3.8–8.9; 9) mm, mature (15–39 days) 10.1 ± 1.0
(8.5–11.5; 15) mm. Maximum body width 390 ± 67
(165–470; 46) [400]. Buccal capsule length 239 ± 21
(163–280; 37) [260], width 216 ± 21 (175–270; 37) [270].
Teeth height 45.4 ± 11.7 (25–80; 20) [60]. Oesophageal
length 965 ± 126 (650–1 275; 33) [900], width 169 ± 32
(70–220; 33) [170]. Copulatory bursa symmetrical
with single dorsal lobe, paired lateral and ventral lobes
(Figs. 10, 11). Paired lateroventral prebursal papillae,
small, anterior to copulatory bursa. Dorsal ray bifurcates
distally with each short stem terminating in 3 digitations.
Externodorsal ray arises proximally from dorsal ray, does
not reach edge of lateral lobe. Lateral rays in contact proximally, separated for distal half; anterolateral ray shorter
than other lateral rays; mediolateral and posterolateral
rays approximately equal in size, tips reach edge of lateral
lobe. Lateroventral and ventroventral rays equal in length
and fused, tips reach edge of ventral lobe. Gubernaculum
elongate, posteriorly clavate; length 104 ± 22 (50–150;
24) [105]. Paired spicules, length 762 ± 47 (660–860; 34)
[670]; juvenile (8–14 days) 748 ± 51 (660–800; 6), mature (15–39 days) 733 ± 39 (670–800; 8). Spicules feature
transverse striations and sharp tips. Genital cone prominent with long, thin, bilaterally paired papillae present
near posterior margin.
Female (description based on 112 specimens; for
detailed morphometric data – see Tables 1, 2): Total
body length 13.5 ± 3.5 (4.5–20.2; 52) [13.3] mm; juvenile (8–14 days) 8.9 ± 1.8 (5.3–11.0; 7) mm, mature
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2
3a
3b
100 μm
1
4b
100 μm
1 mm
4a
5b
100 μm
1 mm
5a
Figs. 1–5. Photomicrographs (a) and line drawings (b) of Uncinaria sanguinis sp. n. from Australian sea lion (Neophoca cinerea).
Fig. 1. Allotype female. Fig. 2. Holotype male. Fig. 3. Holotype male, buccal capsule, dorsoventral view. Fig. 4. Juvenile male, buccal capsule, oblique lateral view. Fig. 5. Female, buccal capsule, en face view.
(15–101 days) 15.0 ± 1.7 (12.4–18.5; 17) mm. Maximum
body width 451 ± 90 (110–590; 52) [520]. Buccal capsule length 287 ± 31 (200–330; 25) [300], width 257 ± 24
(185–300; 25) [265]. Teeth height 54 ± 12 (38–75; 17)
[75]. Oesophageal length 1 059 ± 130 (750–1 275; 25)
[1 000], width 183 ± 37 (90–240; 25) [170]. Tail bluntly
conical, length 204 ± 39 (150–285; 27) [225], terminating with a short spike (Fig. 8). Vulva located in middle to
258
posterior third of body, 5.2 ± 1.6 (1.8–9.8; 29) [5.0] mm
to posterior end; prominent anterior and posterior vulval
lips (Fig. 7). Ovejectors longitudinal; uterine eggs thin
shelled, elliptical, 124 ± 6 × 81 ± 9 (110–135 × 55–95;
19) [123 × 80] (Fig. 9).
T y p e h o s t : Australian sea lion, Neophoca cinerea (Péron),
emaciated and freshly deceased pup, female, 15 days old,
6 kg, collected 1 May 2012 (ID SBDP12–088).
Ahead of print online version
Marcus et al.: Uncinaria sanguinis sp. n. from Neophoca cinerea
6b
7a
7b
9
50 μm
10
8a
100 μm
200 μm
200 μm
6a
8b
11b
100 μm
11a
100 μm
Figs. 6–11. Photomicrographs (a) and line drawings (b) of Uncinaria sanguinis sp. n. from Australian sea lion (Neophoca cinerea).
Fig. 6. Allotype female, anterior end, lateral view. Fig. 7. Allotype female, vulva, lateral view. Fig. 8. Allotype female, tail with
mucron, lateral view. Fig. 9. Ovum. Fig. 10. Holotype male, copulatory bursa and spicules, dorsoventral view. Fig. 11. Male, copulatory bursa and gubernaculum, dorsoventral view with rays spread. Abbreviations: al – anterolateral; d – dorsal; ed – externodorsal;
g – gubernaculum; gp – genital cone papillae; lv – lateroventral; ml – mediolateral; pl – posterolateral; pp – prebursal papillae;
vv – ventroventral.
T y p e l o c a l i t y : Seal Bay (35°59'37''S; 137°18'29''E), Kangaroo Island, South Australia, Australia.
O t h e r l o c a l i t i e s : Seal Bay, Kangaroo Island (35°59'40''S;
137°19'00''E), Dangerous Reef, Spencer Gulf (34°48'54''S;
136°12'43''E), and South Page Island, Backstairs Passage
(35°46'37''S; 138°17'31''E); South Australia, Australia.
S i t e o f i n f e c t i o n : Based on necropsy, adult and juvenile
worms in large numbers in the small intestine of Australian
sea lion pups. Unembryonated eggs released into the environment with faeces.
T y p e s p e c i m e n s : Holotype male (AHC 35806), allotype
female (AHC 35807), and 58 paratypes (AHC 35808 and
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Table 1. Measurements of Uncinaria sanguinis sp. n. collected from Australian sea lion (Neophoca cinerea) pups.
Location
Sex
DR
SPI
Mature ♀
Max. sample size
17
Hosts examined
10
Body length
10.2–20.2 (6.0)
Body width
450–590 (100)
Buccal capsule length 250–330 (30)
Buccal capsule width 235–300 (50)
Teeth height
40–75 (15)
Oesophageal length 1 000–1 150 (40)
Oesophageal width
120–230 (50)
Tail length
160–285 (35)
Vulva to posterior end 3.5–9.8 (0.9)
Average egg length
110–128 (15)
Average egg width
73–95 (7)
Gubernaculum length
Spicule length
-
Seal Bay
1
24
1
13
16.9 10.1–18.7 (4.0)
540 320–580 (140)
255–320 (15)
225–300 (10)
50–75 (-)
- 1 000–1 275 (125)
170–240 (0)
245
150–275 (95)
7.0
4.6–7.4 (1.9)
115–135 (8)
55–88 (33)
-
Seal Bay
Juvenile ♀
DR
SPI
Mature ♂
10
7
4.5–11 (2.2)
110–380 (10)
200–275 (-)
185–275 (-)
38–60 (-)
750–1 000 (-)
90–195 (-)
150–235 (-)
1.8–4.1 (-)
-
14
8
6.6–11.4 (1.4)
350–450 (50)
220–265 (15)
175–225 (35)
35–50 (15)
850–1 050 (150)
125–195 (55)
50–100 (50)
680–850 (110)
Seal Bay
1
20
1
10
10.1 8.5–11.5 (2.0)
460 350–470 (50)
220 215–280 (50)
190 190–270 (40)
40–80 (35)
1 025 900–1 275 (25)
165 160–220 (35)
90–150 (45)
760 670–860 (60)
Seal Bay
Juvenile ♂
11
6
3.8–8.9 (3.8)
165–380 (85)
163–250 (50)
180–250 (25)
25–45 (-)
650–900 (100)
70–180 (50)
75–125 (50)
660–800 (110)
Measurements are given in mm for body length and vulva to posterior end. All other measurements are in µm. Reported values are minimum–maximum. Values in parentheses are the maximum range observed within individual hosts. Abbreviations: DR – Dangerous Reef ; SPI – South Page Island.
Table 2. Comparative measurements (mean ± standard deviation) of body and spicule lengths for Uncinaria sanguinis sp. n.
collected from Australian sea lion (Neophoca cinerea) pups of known age.
Body length (mm)
Pup age (days)
n
♀
Juvenile
8
11
12
14
1
2
2
2
Mature
15
27
29
37
39
101
4
4
3
2
2
2
Spicule length (µm)
n
♂
n
♂
5.25
9.70b ± 0.42
8.75b ± 0.35
9.90b ± 1.56
3
2
2
2
5.48 ± 1.89
8.12b ±0.18
8.25b ± 0.00
7.94b ± 1.33
2
2
2
790 ± 14
740 ± 28
715 ± 78
13.18c ± 0.64
16.38d ± 1.49
14.33ce ± 0.26
14.88cde ± 1.77
16.38de ± 0.53
15.75de ± 2.83
4
3
4
2
2
-
9.12bc ± 1.01
10.52c ± 0.96
9.95bc ±0.77
10.62c ± 0.53
11.00c ± 0.35
-
2
2
3
1
-
735 ± 92
735 ± 21
723 ± 25
750
-
a
a
For each sex, means that do not share a superscript letter (a–e) are significantly different (P < 0.05).
AHC 46823–46850) deposited in the Australian Helminthological Collection of the South Australian Museum, Adelaide, Australia. Additional paratypes deposited in the
U.S. National Parasite Collection, Maryland, USA (USNPC
107521–107522; n = 20), Natural History Museum, London,
UK (NHMUK 2013.11.13.1–2013.11.13.20; n = 20), and
Helminthological Collection of the Institute of Parasitology,
České Budějovice, Czech Republic (IPCAS N–1047; n = 2).
DNA sequences (GenBank accession numb e r s ) : Partial 28S rDNA: KF690639–KF690649; ITS1,
5.8S rDNA, ITS2: KF690650–KF690670.
E t y m o l o g y : The species epithet reflects the haemorrhage
and effective exsanguination caused by large burdens of this
parasite in the type host. The name is derived as Latin singular genitive.
Morphometrical variation. Measurements were obtained for examined features with inter-host morphometrical variation being greater than intra-host variation (Ta-
260
ble 1). Ranges of all morphometrical features overlapped
for mature hookworms from Seal Bay, Dangerous Reef
and South Page Island. Juvenile hookworms, collected
from pups at Seal Bay with prepatent infections, differed
from mature specimens by the absence of eggs in females
and generally reduced magnitude for morphometrical
values, however, no functional morphological differences
were identified (Fig. 4).
Host age and hookworm sex significantly influenced
hookworm body length (F8, 29 = 3.89, P = 0.003), with significant sexual dimorphism, associated with maturity, observed from 15 days (females longer than males, P < 0.05;
Table 2). Whilst body length generally increased with host
age and mature females were significantly longer than juvenile females (P < 0.05), males at 15 and 29 days were
not significantly longer than juvenile males (P > 0.05).
Additionally, spicule length was not significantly related
to host age (F6, 7 = 0.49, P = 0.796).
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Marcus et al.: Uncinaria sanguinis sp. n. from Neophoca cinerea
12
13
Figs. 12, 13. Phylogenetic relationships of host-associated Uncinaria spp. estimated with concatenated ITS1, ITS2, and 28S rDNA
sequence data. Fig. 12. Maximum Likelihood tree based on the K2 model in MEGA5 with the highest log likelihood (-2117.3390).
Numbers above the nodes represent bootstrap values (1 000 replicates), values below the branches represent Bayesian posterior
probabilities based on the K2 model calculated in MrBayes. The tree is drawn to scale, with branch lengths measured in the number
of substitutions per site. Fig. 13. The majority consensus rule tree constructed using Maximum Parsimony based on SPR algorithm
in MEGA5. Numbers above the nodes represent bootstrap values (1 000 replicates). The multiple sequence alignment included nine
nucleotide sequences and a total of 1 149 positions in the final dataset. Abbreviations: AF – Arctic fox, Alopex lagopus; AFS – Australian fur seal, Arctocephalus pusillus doriferus; ASL – Australian sea lion, Neophoca cinerea; CSL – California sea lion, Zalophus californianus; MMS – Mediterranean monk seal, Monachus monachus; NZSL – New Zealand sea lion, Phocarctos hookeri;
NFS – northern fur seal, Callorhinus ursinus; SASL – South American sea lion, Otaria byronia; SAFS – South American fur seal,
Arctophoca australis; SES – southern elephant seal, Mirounga leonina; SSL – Steller sea lion, Eumetopias jubatus.
DNA sequences. Four DNA markers were amplified.
The complete ITS1 of U. sanguinis was 364 bp long, ITS2
was 227 bp long and the 5.8S rDNA sequence of U. sanguinis was 153 bp long (Seal Bay, n = 10; Dangerous Reef,
n = 10; South Page Island, n = 1). The partial 28S rDNA
sequence of U. sanguinis was 992 bp long (Seal Bay,
n = 5; Dangerous Reef, n = 5; South Page Island, n = 1).
Molecular distance analysis. Uncinaria sanguinis
from Seal Bay, Dangerous Reef and South Page Island
demonstrated 100% sequence similarity at ITS1, ITS2,
5.8S rDNA, and 28S rDNA. There was a single polymorphism (C/T) in ITS1 at position 199 in one worm (AHC
46824; KF690651), which was not present in another
worm from the same host (AHC 46825; KF690652). The
polymorphism was confirmed by sequencing PCR product generated using two different DNA polymerases. The
historical Australian sea lion and New Zealand fur seal
hookworm ITS sequences (HE962176 and HE962175, respectively) demonstrated 100% similarity to U. sanguinis
sequences. Additionally, the New Zealand fur seal hookworm ITS sequence (HE962175) demonstrated an ambiguous nucleotide at position 199 in ITS1, corresponding to
the polymorphic site in worm AHC 46824 (KF690651).
Pairwise comparisons with Uncinaria from other otariid,
phocid, canid and felid hosts demonstrated high levels of
sequence similarities at ITS1 and ITS2 (Table S1). Fewer
differences were evident between hookworm species at
the 28S rDNA locus; sequence similarity to U. sanguinis
was 99.3–99.8% (Table S2). The 5.8s rDNA locus was
invariant across all Uncinaria species.
Molecular character analysis. Bayesian analysis
demonstrated 100% support (posterior probability) for
one phocid and three otariid hookworm-clades (Figs. 12,
14). The phocid clade consists of two sequences, representing two putative new species, one each from the
southern elephant seal and the Mediterranean monk seal.
The otariid clade is further divided into the North and
South American clades and the Oceanic clade (Figs. 12,
14). The North American otariid clade contains sequences
belonging to U. lucasi from the northern fur seal and the
Steller sea lion, and sequences belonging to the putative
new species Uncinaria ‘species A’ sensu Nadler et al.
(2000) from the California sea lion. The South American
otariid clade consists exclusively of sequences belonging
to U. hamiltoni from the South American sea lion and
the South American fur seal. The Oceanic otariid clade
consists of sequences belonging to U. sanguinis from
the Australian sea lion and two sequences from unnamed
Uncinaria spp. from the New Zealand sea lion and the
Australian fur seal, respectively. Hookworms from the
New Zealand fur seal are provisionally considered to be
U. sanguinis sp. n. on the basis of limited data.
Maximum Likelihood and Maximum Parsimony analyses provided 100% bootstrap support for separate phocid
and otariid hookworm-clades (Figs. 12–14). The Maximum Likelihood analysis demonstrated 100% support for
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Fig. 14. Schematic of phylogenetic relationships of pinniped Uncinaria spp. constructed from the majority consensus of Bayesian
inference, Maximum Likelihood and Maximum Parsimony methods with concatenated ITS1, ITS2, and 28S rDNA sequence data.
Uncinaria felidis and Uncinaria sp. from Arctophoca forsteri are excluded due to incomplete data. The bars indicate nucleotide
character changes with reference to the outgroup Uncinaria stenocephala. Homoplastic characters shared with U. stenocephala are
indicated by white bars with the derived character state at this position indicated in parentheses. Nucleotide gaps are indicated by ‘#’.
Alignment positions are specific to each locus (a – ITS1; b – ITS2; c – 28S rDNA). Four clades are evident, one from phocid hosts
and three from otariid hosts. three otariid hookworm-clades and moderate (67%) support for the three sequence lineages within the Oceanic
clade (Fig. 12). The Maximum Parsimony analysis moderately (61%) supported the South American and Oceanic
262
clades and, in contrast to the other phylogenetic analyses, provided only weak (< 50%) support for separate
sequences within the Oceanic clade (Fig. 13). However,
fixed rDNA character state changes provide evidence for
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Marcus et al.: Uncinaria sanguinis sp. n. from Neophoca cinerea
independent evolutionary lineages and species delimitation (Fig. 14).
Differential diagnosis. Uncinaria sanguinis demonstrates the general morphological characteristics of the
Ancylostomatinae and the specific features of the genus
Uncinaria, including the dorsally directed anterior extremity, a globular buccal capsule with continuous walls,
well-developed cutting plates, and a dorsal gutter (Lichtenfels 2009). The species can be differentiated from
all other named species in the genus Uncinaria on the
basis of morphological features and available molecular
data. Morphologically, U. sanguinis is very similar to
the otariid hookworms U. lucasi and U. hamiltoni, demonstrating subtle morphological differences. An annular
thickening of the base of the buccal capsule, observed in
U. sanguinis and U. hamiltoni, was reported absent in the
description of U. lucasi, but subsequently noted in other
specimens (Lyons and DeLong 2005). Male specimens
of U. sanguinis exhibit shorter anterolateral rays relative
to the other lateral rays, a feature shared with U. hamiltoni, differentiating these species from U. lucasi which
exhibits lateral rays of almost equal length (Baylis 1947).
The relatively shorter externodorsal rays of U. sanguinis
distinguish it from U. hamiltoni – see Baylis (1933).
Female specimens of U. sanguinis are morphologically
indistinguishable from U. lucasi and U. hamiltoni. Uncinaria sanguinis is definitively identified and delimited
from U. lucasi by thirteen fixed character state changes at
three loci (ITS1, n = 3; ITS2, n = 6; 28S, n = 4) and from
U. hamiltoni by seven changes at two loci (ITS1, n = 5;
28S, n = 2).
The size of eggs from U. sanguinis is similar to those
from U. lucasi and U. hamiltoni, and is approximately
twice the size of eggs from Uncinaria spp. parasitic in
terrestrial mammals, differentiating female otariid hookworms. The only other native Australian hookworm, Uncinaria hydromyidis Beveridge, 1980, described from the
Australian water rat, Hydromys chrysogaster Geoffroy,
a murid host in north-eastern Queensland, may be further
distinguished from U. sanguinis by its relatively longer
ventral and dorsal rays, blunt spicule tips, the absence of
the annular buccal capsule thickening, and female specimens feature inconspicuous vulval lips (Beveridge 1980).
Uncinaria bidens (Molin, 1861), found in procyonids
(Carnivora), is differentiated from U. sanguinis by relatively longer ventral rays and the angulation of the lateral
ray tips (Olsen 1968).
The felid hookworms, U. felidis and Uncinaria maya
Hasegawa, 1989, differ from U. sanguinis by their posterolateral, externodorsal and dorsal rays being of equal
length and female specimens exhibit prevulvar flaps or
protruding anterior lips (Maplestone 1939, Hasegawa
1989). Uncinaria bauchoti Chabaud, Brygoo et Tchéprakoff, 1964 and Uncinaria olseni Chabaud et DuretteDesset, 1975, identified from tenrecid (Afrosoricida)
and tupaiid (Scandentia) hosts, respectively, exhibit
large deirids and have been considered by some authors
(Chabaud et al. 1966, Lichtenfels 2009) to represent
a subgenus, Megadeirides Chabaud, Bain et Houin, 1966.
All other Uncinaria species, found in carnivore hosts
from the ailurid, canid, mustelid and ursid families, may
be morphologically discriminated from U. sanguinis and
each other on the basis of their lateral rays using Olsen’s
(1968) key. Molecularly, U. sanguinis is delimited from
U. stenocephala by 54 fixed character state changes at
three loci (ITS1, n = 27; ITS2, n = 24; 28S, n = 3) and
from U. felidis by 32 changes in ITS2.
DISCUSSION
Hookworms collected from Australian sea lions were
found to belong to a single novel species, Uncinaria
sanguinis sp. n. Morphological and molecular investigations did not discriminate between specimens from three
South Australian colonies and provided no evidence for
the presence of cryptic species or geographical variants.
This is the third species within the genus Uncinaria to be
described and named from otariid hosts and is morphologically most similar to U. lucasi and U. hamiltoni. Differences in the relative lengths of the bursal rays differentiate these species and fixed rDNA sequence diversity
demonstrates independent evolutionary lineages.
The potential for host-induced effects on parasite morphology engenders caution when deciding whether we
have new species (George-Nascimento et al. 1992, PérezPonce de León and Nadler 2010). As such, assessing the
normal range of morphometric variation within a species
is of critical importance for accurate species-description
and comparisons. We found little intra-host variation for
specimens of U. sanguinis from Australian sea lions. Similarly, Olsen and Lyons (1965) reported no variation in
the size of U. lucasi within individual northern fur seals.
These findings support the hypothesis that pinnipeds acquire hookworm infection over a short period of time,
likely via the transmammary route shortly after birth
(Olsen and Lyons 1965). However, we also observed
large morphometric variation between hosts, in part related to host age, highlighting the importance of examining
specimens from multiple host-individuals across a range
of ages in order to assess the extent of species variation.
Interestingly, juvenile hookworms demonstrated no functional morphological differences when compared to adult
hookworms and may be associated with host-pathology,
which has implications for the detection of disease in live
animals.
The wide morphometric ranges we obtained overlap
with those of other Uncinaria spp., suggesting limited
utility for quantitative morphometrics for species discrimination within this genus. Similar conclusions were
reached by other authors who identified significant hostassociated morphometric differences within U. hamiltoni
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from the South American sea lion and South American fur
seal (George-Nascimento et al. 1992) and within U. lucasi from the northern fur seal and Steller sea lion (Olsen
1952, Nadler et al. 2013). Whether these differences were
host-induced or due to age-dependent variation is unclear
due to limited or unreported host sample sizes and unknown host ages. Regardless, these findings support the
need to employ molecular techniques to delimit and describe morphologically-similar species.
Hookworms from the Australian fur seal, New Zealand
fur seal, and New Zealand sea lion cluster with U. sanguinis within the Oceanic clade. Morphological differences between Oceanic hookworms have not been described
although molecular differences delineate two species
from the Australian fur seal and New Zealand sea lion,
respectively, in addition to U. sanguinis from the Australian sea lion. Hookworms from the New Zealand fur seal
are thus provisionally considered to be U. sanguinis. An
alternative viewpoint may be that all hookworms within
the Oceanic clade are U. sanguinis and that molecular
differences reflect intraspecific or geographical variation
(Ramos et al. 2013). Whether U. sanguinis demonstrates
host-specificity or is capable of infecting other hosts remains to be tested. Investigation of hookworm population
structure at DNA markers subjected to greater rates of
substitution, such as mitochondrial DNA, may demonstrate the level of genetic interchange between colonies
and host-associated hookworms and clarify their taxonomic identities further (Gasser and Newton 2000).
Recognising, describing and identifying parasites of
free-ranging wildlife species are critical processes for the
comprehensive investigation and management of associated disease (Thompson et al. 2010). Delimiting parasitic
species is essential for examining host-parasite-environment-anthropogenic interactions, implementing and moni-
toring management programs, and ensuring the conservation of parasites and their hosts. Hookworm infection is
a recognised cause of morbidity and mortality in otariid
hosts (Castinel et al. 2007, Chilvers et al. 2009, DeLong et
al. 2009, Spraker and Lander 2010). In this study, we coupled morphological analysis with molecular techniques to
describe and identify a novel species of hookworm in the
Australian sea lion. This work contributes towards resolving the taxonomic uncertainty within the genus Uncinaria
and provides critical data regarding an important pathogen
of an endangered mammal. Further investigation and formal identification of the species of hookworms parasitising other pinniped hosts is recommended.
Acknowledgements. We thank the staff at Seal Bay, Department
of Environment, Water and Natural Resources for field assistance and the collection of deceased pups, in particular Clarence
Kennedy and Janet Simpson; Liisa Ahlstrom, Loreena Butcher,
Michael Edwards, Simon Goldsworthy, Claire Higgins, Janet
Lackey, Zoe Larum, Theresa Li, Andrew Lowther, Paul Rogers, Laura Schmertmann, Adrian Simon, Ryan Tate, Michael
Terkildsen, Mark Whelan, Peter White, Sy Woon, and Mariko
Yata for field assistance; Denise McDonell of The University of
Sydney for parasite morphology and laboratory assistance; Benjamin Haynes of The University of Sydney for performing the
DNA extractions; Carsten Minten of Olympus Australia for use
of equipment and assistance with microphotography; Ian Beveridge of The University of Melbourne for helpful comments
on hookworm morphology and presentation; and Glenn Shea
of The University of Sydney for assistance with Latin grammar.
This work was supported by the Australian Marine Mammal
Centre, Department of the Environment, Australian Government (grant number 09/17), Winifred Violet Scott Foundation
(2007), and the Whitehead Bequest Conservation 2013, Faculty
of Veterinary Science, The University of Sydney. We thank the
anonymous reviewers and Tomáš Scholz for their constructive
comments on the manuscript.
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Received 1 November 2013
Accepted 30 January 2014
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